sciences dissertation

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Introduction

Environmental pollution by metals is a serious threat today due to increased industrial revolution and has become extensive. The current worldwide mine production of various heavy metals such as Cu, Cd, Pb, and Hg is very high (Pinto et al., 2004). These heavy metal sources include a number of diverse anthropogenic sources (textile, tannery and other industrial effluents, industrial solid wastes, urban runoff, sewage treatment plants, boating activities, agricultural fungicide runoff, domestic garbage dumps, and also by mining operations). Metal toxicity and its effect on the growth and metabolism in higher plants is a subject that has a wide economical and ecological interest and also have been widely reviewed and gaining importance on several occasions over the last few decades (Brown and Jones, 1975; Foy et al., 1978; Ernst et al., 1992; Das et al., 1997; Sanitá di Toppi and Gabrielli, 1999; Hall, 2002; Clemens et al., 2002).

Another interesting reason for this is out of the ninety naturally occurring elements fifty-three are heavy metals (Weast, 1984). Few metals are found to be essential for growth of plants, while few are found to be toxic for the growth of plants. Some of the heavy metals don't interact with the plant metabolism directly but decreases the level of soil microbes, affecting the growth of the plants (Niess, 1999). However, the accumulation in the soil has become a worldwide problem leading to the loss of agricultural productivity (Hasan, 2007).

Among the heavy metals Cd is of special concern because of its potential toxicological profile even at lower concentration (Das & Samantray, 1997). Cadmium is a toxic trace pollutant for humans, animals and plants, which enter the environment from various industrial processes, phosphate fertilizers and other anthropogenic activities, which then enters the food chain (Wagner, 1993). It has been estimated that more than 45% of all cadmium is used in electroplating, and that of 10% of total industrial cadmium use ends up in wastewater streams.

Interests in the ecological effects of heavy metals has been growing recently, and assessing the phytotoxicity of cadmium is one of the most important criteria being used to avoid environmental risks, before these effluents can be recycled back for agricultural irrigation after some remediation or treatment processes. So it is inevitable that we should cope with the environmental damages induced by increased concentration of cadmium.

The removal of cadmium (II) from wastewater stream therefore is of great importance as it can directly contaminate the ground water, aquatic organisms, agricultural lands and also the productivity. The global threat of reduction of uncontaminated ground water is also increasing exponentially with the scientific, industrial and technological developments (Tchobanoglous et al., 1991). Although various other techniques such as precipitation are most commonly used, to clean up the Cd contaminated water, the drawbacks of using those techniques, increases the need for an alternative technique which is more effective, reliable and also economically viable (USEPA, 1984).

Cadmium being a highly toxic metal pollutant of soil inhibits root and shoot growth and yield production, affects nutrient uptake and homeostasis, and is frequently accumulated by agriculturally important crops including soybean with a significant potential to impair animal and human health (Sanita di toppi and Gabrielli, 1999). In plants such as pea, a number of toxic effects of Cd on metabolism has been reported, such as decreased uptake of nutrient elements (Sandalio et al., 2001), inhibition of various enzyme activities (Obata et al., 1996) and induction of oxidative stress (Lozano-Rodriet al., 1997, Romero-Puertas et al., 1999, Sandalio et al., 2001) including alterations in enzymes of antioxidant defense system (Dalurzo et al., 1997, Sandalio et al., 2001). However only little is known about the biochemical mechanisms underlying the intraspecific variation in the sensitivity versus tolerance to Cd in plants (Ashraf et al., 2005).

Understanding the connections between a plant's initial responses and the downstream events that constitute successful adjustment to its altered environment is one of the next grand challenges of plant biology (Ruth Grene, 2002). One of the common characteristics of these heavy metals such as cadmium is the induction of oxidative stress generating active oxygen species (AOS). Plants have evolved various protective defense mechanisms to eliminate or reduce these AOS species. One of them is the enzymatic or non-enzymatic antioxidant system, including peroxidase, Super oxide dismutase, catalase, glutathione peroxidase etc. Each of these parameters has physiological function under non-stressed conditions, but their activity or quantity is increased under oxidative stress (Sara Erdei, 2002).

This study becomes more important because legume crops are less tolerant to Cd toxicity than cereals, grasses and frequently encounter strong inhibition of biomass production even at the less than micro molar range of cadmium (Inouhe et al., 1994). No much of the studies have been reported in the effect of cadmium in reproductive stage of soybean plant. Especially studies on the response of plants can very much help in increasing productivity, improving biomass, and reducing the green house gases, and also in the identification of Cd tolerant plants. Research studies on Cd effect on the economically important soybean crop may also be helpful to meet the increasing demand for soybean in India. The increasing global demand for soybean, oil and other products, rise in price at the international level particularly after the usage of soybean for biodiesel production, made us to select soybean as the plant of our research interest in this study.

With the increasing scarcity for water and increasing pollution of ground water the irrigation of the legumes has become a big problem, in addition to the contaminated and less fertile soil for the farmers. Hence it is very essential to derive mechanisms to clean up technologies to remove the contaminants form wastewater and reuse of it for irrigation of legumes. Activated carbon absorption has been used successfully for the advanced treatment of municipal and industrial wastewater. Adsorption occurs when molecules adhere to the internal walls of pores in carbon particles produced by thermal or acid chemical activation (US EPA, 2000).

Production of activated carbon from an easily available agro waste makes this technique more apt to adapt in a large-scale level. Also the reduction in the production cost by modification of already available techniques is very important and makes this process economically very feasible. Hence in our study we have selected the agricultural waste, shell from Anacardium occidentale L., a member of the Anacardiaceaefamily, of which the cashew is by far the most important economically. Anacardium occidentale L., is cultivated commercially in 32 countries on over 7.5 million acres. Cashew is now the #1 tree nut crop in the world, since its production surpassed that of almond in 2003.

Average yields worldwide are about 600 lbs/acre. And India ranks second in its production contributing to 25% of the total production worldwide. Anacardium occidentale L produce 2 additional products of commercial value from their fruit: cashew apples and cashew nut shell liquid. After the CNSL is taken, the left out biological waste shell is thus considered for the production of low cost activated carbon in our study, which is available in higher quantity at a very cheaper price. Hence this study may be increasing one more commercially important product from the waste of Anacardium occidentale L.

Objectives of The Study

The major objectives of this research project are

Use of the left out agro waste shell of Anacardium occidentale L., for the production of low cost activated carbon, in turn increasing one more commercially important product from the waste of Anacardium occidentale L., this also may be helping to improve the production of activated carbon in India to meet the increasing global need in the activated carbon industry.

Development of an activated carbon process as the cleaning technology for removing heavy metal cadmium, from the Cd contaminated water

Reuse of the treated water for growing economically important legume soybean and studying the response of soybean grown with the cadmium contaminated water before and after treatment.

Specific Aims

The specific aims of this study are as follows

Study of the effect of Cd on the growth, stress and antioxidant metabolism of Glycine max (L.) Merr. Plants by petriplate and pot culture studies.

Production and characterization of low cost activated carbon from the agro waste nut shell of Anacardium occidentale L.

Batch mode experiments for the optimizing the conditions for maximum removal of Cd by the activated carbon from the biological waste nut shell of Anacardium occidentale L.

Application of the treated water for irrigation and study of effect of Cd contaminated water after treatment on the growth, stress and antioxidant metabolism of Glycine max (L.) Merr. Plants by petriplate and pot culture studies.

Evaluation of the effect of treated water on the growth on the growth of soybean over the treated water.

A study on uptake and localization of cadmium by various parts of soybean

Experimental Design and Salient Findings

The experimental protocols are deduced and the work is designed in order to fulfill the objectives of the study and was carried out in eight phases, following standard protocols wherever necessary.

PHASE-I:

Collection of Seeds, Soil and Biological Waste Sample

A. COLLECTION OF SEEDS, SOIL AND AGRO WASTE:

Co-2 variety soybean [Glycine max (L.) Merr] seeds were collected from Millets and New Area Farm, Centre for Plant Breeding and Genetics, Tamilnadu Agricultural University, Coimbatore, and used for the study. The biological waste of shell of Anacardium occidentale L.was collected and the activated carbon is produced by the acid activation process.

B. PREPARATION OF SYNTHETIC EFFLUENTS:

The synthetic effluents with concentrations varying from 5, 10, 15, 20 and 25 ppm concentration of cadmium were prepared by using cadmium chloride, by calculating the level of cadmium in CdCl2 and used for growing the plants during germination, pot culture studies and for Cd adsorption studies. Distilled water is used as the control for all the experiments.

C. COLLECTION OF THE SOIL AND SOIL STUDY

Red soil was collected from the agricultural fields of Coimbatore, the composition is checked, and used throughout the study. The soil parameters checked includes,

Findings:

The above parameters were checked to ensure that the soil is suitable for the growth of soybean. The Cd level was determined using Atomic Absorption Spectroscopy and found to be below non detectable limit and hence used for the study.

FINDINGS:

In the observations made with five plants in each group on 20th day, the following results were observed. The growth parameters such as root length, shoot length, plant height, relative root length percentage, relative shoot length percentage, tolerance index, no of leaves, trifoliates, fresh weight, dry weight were found to be decreased significantly with increasing concentrations of cadmium level in the effluents when compared to control. Also dry mass/fresh mass ratio, stem mass ratio, root mass ratio were found to be increased, while the other parameters such as leaf mass ratio, total dry matter yield under cadmium stress, percentage weight gain, seed vigor index, leaf area measurements, leaf area index, leaf weight fraction, specific leaf area, leaf area ratio were also found to be decreasing when compared to control group plants, with increasing levels of cadmium in the effluents.

FINDINGS:

In the observations made with five plants in each group on 40th day, the following results were observed. The growth parameters such as root length, shoot length, plant height, relative root length percentage, relative shoot length percentage, tolerance index, number of leaves, trifoliates, nodules per plant, fresh weight, dry weight of individual organs, and whole plant, leaf mass ratio, total dry matter yield under cadmium stress, percentage weight gain, seed vigor index, leaf area measurements, leaf area index, leaf weight fraction, specific leaf area and leaf area ratio were found to be decreased significantly. The dry mass/ fresh mass ratio, stem mass ratio and root mass ratio were found to be increasing with increase in the concentration of cadmium. The growth parameters connecting the observations made on 20th and 40th day observations such as absolute growth rate, crop growth rate, unit leaf rate, net assimilation rate, relative growth rate, leaf area duration values showed a significant difference from the values of control group plants.

III.C. BIOCHEMICAL PARAMETERS ESTIMATED ON 20TH DAY IN ROOT AND SHOOT TISSUES:

FINDINGS:

The levels of growth parameters such as Chlorophyll-a, b and total Chlorophyll were significantly found to be affected with the increase in the cadmium concentrations in the effluents. The levels of Total Carbohydrate, Starch and Total Protein were also estimated in root and leaf tissues and were found to be significantly decreased with increasing concentrations of cadmium.

The decreasing levels of Amylase, Glucose-6-phosphate dehydrogenase were also observed with increasing concentrations of cadmium, when compared to control in both root and leaf tissues.

The stress markers like Total Free Amino Acids, Proline and Thiobarbituricacid reactive substances (TBARS) were also found to be increased with increase in the concentrations of cadmium in both leaf and root tissues.

The non enzymatic antioxidants such as Vitamin E, Ascorbic acid, and Reduced Glutathione were found to significantly varying with the varying concentrations of cadmium.

The enzymatic antioxidants such as Catalase, Peroxidase, Glutathione Reductase, Glutathione peroxidase were found to be decreased in their levels with increasing concentration of cadmium.

Superoxidedismutase was found to be increasing with increase in the Cd concentration.

PHASE-IV - PRODUCTION AND USE OF LOW COST ACTIVATED CARBON FOR REMOVAL OF CADMIUM FROM CONTAMINATED EFFLUENT - ADSORPTION STUDIES.

COLLECTION OF THE BIOLOGICAL WASTE.

ACTIVATED CARBON PRODUCTION BY ACID ACTIVATION.

PHYSICAL PROPERTIES OF ACTIVATED CARBON

FTIR ANALYSIS OF ACTIVATED CARBON BEFORE ADSORPTION

SEM ANALYSIS BEFORE ADSORPTION

FINDINGS:

The agricultural waste, shell from Anacardium Occidentale L., were collected, and the activated carbon is produced, the activated carbon of size less than 125 micron was selected and its physical parameters were determined, and is found to be suitable for removal of cadmium from waste water. Fourier Transform Infra Red spectroscopy and Scanning Electron Microscopy analysis were done for the prepared activated carbon.

PHASE-V - BATCH MODE ADSORPTION STUDIES

Batch mode adsorption studies were done for the following parameters

EFFECT OF EQUILIBRIUM TIME

EFFECT OF pH

EFFECT OF VARYING CARBON CONCENTRATION

EFFECT OF VARYING CADMIUM CONCENTRATION

EFFECT OF TEMPERATURE

FINAL PERCENTAGE REMOVAL OF CADMIUM FROM VARIOUS CONC OF Cd SOLUTIONS.

FTIR ANALYSIS OF ACTIVATED CARBON AFTER ADSORPTION

SEM ANALYSIS AFTER ADSORPTION

FINDINGS:

The batch mode experiments were performed for adsorption studies and the optimum equilibrium time, optimum pH, effects of varying carbon concentration and varying carbon concentration and temperature were done and the carbon to cadmium ratio was determined. The final percentage of cadmium removed successfully after treatment with optimum concentrations was determined using Atomic Absorption Spectroscopy. The carbon collected after adsorption was analyzed using Fourier Transform Infra Red spectroscopy and Scanning Electron Microscopy.

FINDINGS:

The following observations were made with five plants in each group grown with treated effluents, on 20th day:

The growth parameters such as root length, shoot length, plant height, relative root length percentage, relative shoot length percentage, tolerance index, number of leaves, trifoliates, fresh weight, dry weight were found to be decreased when compared to control. But a highly significant improvement was seen in all these parameters, when compared with the values of plants grown with effluents before treatment. Also dry mass/fresh mass ratio, stem mass ratio, root mass ratio were also found to be varied than the before treatment plants. The other growth parameters such as leaf mass ratio, total dry matter yield under cadmium stress, percentage weight gain, seed vigor index, leaf area measurements, leaf area index, leaf weight fraction , specific leaf area, leaf area ratio were also found to be improved when compared to before treatment plants, although it is decreasing when compared to control group plants.

FINDINGS:

The levels of growth parameters such as Chlorophyll-a, b and Total Chlorophyll were significantly found to be improved with the increase in the cadmium concentrations in the effluents after treatment. The levels of Total Carbohydrate, Starch and Total Protein were also found to be increased significantly when compared to before treatment plants in both root and leaf tissues. Although a decreasing levels of Amylase, Glucose-6-phosphate dehydrogenase were observed with increasing concentrations of cadmium, when compared to control, a significant increase in their levels was observed when compared with the plants grown before treatment in both root and leaf tissues.

The stress markers like Total Free Amino Acids, Proline and Thiobarbituric Acid Reactive Substances(TBARS) were also found to be decreased in both leaf and root tissues when compared with the plants grown using effluents before treatment.

The non enzymatic antioxidants such as Vitamin E, Ascorbic acid and Reduced Glutathione were found to significantly varying with the varying concentrations of cadmium after treatment.

The enzymatic antioxidants such as Catalase, Peroxidase, Glutathione Reductase, and Glutathione Peroxidase were found to be significantly increased in the plants grown after treatment. Also level of Superoxide dismutase was found to be reduced than the level found in plants grown using effluents before treatment.

PHASE-VIII - UPTAKE AND LOCALIZATION OF CADMIUM BY SOYBEAN PLANTS

BEFORE TREATMENT

The level of cadmium uptake was measured by atomic absorption spectrophotometer in the tissues of root, shoot, leaves, and pod of soybean grown using Cd contaminated water before treatment.

AFTER TREATMENT

The level of cadmium uptake was measured by atomic absorption spectrophotometer in the tissues of root, shoot, leaves, and pod of soybean grown using Cd contaminated water after treatment.

FINDINGS:

The uptake and localization of cadmium levels and correlated to the results obtained in biochemical and physical parameters.

Conclusions

The results obtained from the present study indicates

The effect of Cd on the growth, stress and antioxidant metabolism of Glycine max (L.) Merr. Plants by petriplate and pot culture studies were studied and are found to be significant.

The production of low cost activated carbon from agro waste of Anacardium occidentale L.., and the removal of Cd from the waste water by the low cost AC are significant.

Use of the treated water for irrigation and its effect on the growth, stress and antioxidant metabolism of Glycine max (L.) Merr. Plants by petriplate and pot culture were studied. The results may be helpful in better understanding of the various growth, metabolic and defense response mechanisms of the Glycine max (L.) Merr. plants, grown with Cd contaminated water before and after treatment using activated carbon.

The effect of treated water on the growth over the non-treated water was evaluated and the results show the efficiency of the treated Cd contaminated waste water for irrigation.

The results also suggest that this method may be useful as a secondary treatment method in the water treatment plants involved in treatment of sewage wastewaters.

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